Neural Progenitor Cell Differentiation

ABSTRACT

Differentiation and stability of neural stem cells can be enhanced by in vitro or in vivo culturing with one or more extracellular matrix (ECM) compositions, such as collagen I, IV, laminin and/or a heparan sulfate proteoglycan. In one aspect of the invention, adult mammalian enteric neuronal progenitor cells can be induced to differentiate on various substrates derived from components or combinations of neural ECM compositions. Collagen I and IV supported neuronal differentiation and extensive glial differentiation individually and in combination. Addition of laminin or heparan sulfate to collagen substrates unexpectedly improved neuronal differentiation, increasing neuron number, branching of neuronal processes, and initiation of neuronal network formation. In another aspect, neuronal subtype differentiation was affected by varying ECM compositions in hydrogels overlaid on intestinal smooth muscle sheets. The matrix compositions of the present invention can be used to tissue engineer transplantable innervated GI smooth muscle constructs to remedy aganglionic disorders.

RELATED APPLICATIONS

This application is a Divisional Application of Ser. No. 14/216,391,filed Mar. 17, 2014, which claims priority of U.S. ProvisionalApplication No. 61/788,285, filed Mar. 15, 2013, which is herebyincorporated in its entirety by reference.

GOVERNMENT RIGHTS

This invention was made with U.S. government support by the NationalInstitutes of Health Grant Nos. RO1DK071614 and RO1DK042876. The U.S.government has certain rights in the invention.

TECHNICAL FIELD

The field of this invention is tissue engineering and, in particular,re-innervation of organs and body tissue.

BACKGROUND

An uninterrupted enteric nervous system with the preservation ofmyenteric ganglia is required for intestinal motility and function.Motor neurons of the myenteric ganglia predominantly expressacetylcholine/tachykinins (excitatory) or nitric oxide/inhibitorypeptides/purines (inhibitory) to mediate smooth muscle contraction andrelaxation. Partial, selective, or total loss of nerve function and/orloss of nerve cell populations within organs and other body structuresare characteristic of numerous diseases and disorders. For example,aganglionosis of various lengths of distal gut is the central pathologyin Hirschsprung's disease. Enteric neuropathy is also secondary toseveral other disorders (e.g., diabetes, Parkinson's disease, andinflammation) resulting in gastrointestinal dysfunction.Gastrointestinal motor function is controlled by the intramural entericnervous system. It is a complex interplay between the smooth muscle ofthe muscularis externa and the two enteric neuronal plexi.

Neural stem cell therapy is an emerging therapy that aims to reinstateneuronal function and thus gastrointestinal motor function byrepopulating the enteric plexi. The research is driven by twosignificant findings: i) neural stem cells can be isolated from adultmammalian gut, including the ganglionated colon of Hirschsprung'spatients; and ii) neural stem cells can be induced to differentiate intoseveral neuronal subtypes and glia characteristic of the enteric nervoussystem (ENS) upon transplantation into explant cultures ofaganglionic/aneural gut, or in vivo into distal colo-rectums in animalmodels.

While neural-crest derived enteric neural stem cells have been isolatedfrom adult mammalian guts, including ganglionic bowel of patients withHirschsprung's disease, there is little information or understanding ofmicroenvironment-driven differentiation, and only limited studiesdescribing subsequent functional behavior of these differentiatedneurons in vitro. Moreover, restoration of nerve functions by neuralstem cell transplantations as a treatment for nerve-loss associateddisorders has not yet been clinically demonstrated, and there exists aneed for methods and materials to support neuroglial cells, and toensure phenotypic stability and long term survival of transplanted orimplanted neural stem cells.

SUMMARY

It has been discovered that extracellular matrix (ECM) compositions canmodulate neural stem cell fate and direct differentiation. The term“extracellular matrix” or “ECM” is used herein to denote compositioncomprising one or more of the following: collagen I, collagen IV,laminin, heparan sulfate, or fragments of one or more of such proteins.

One aspect of the invention includes a method of biasing neural stemcell differentiation having the steps of obtaining a population ofsmooth muscle cells, culturing the smooth muscle cells to form auniaxially-aligned smooth muscle sheet, obtaining a population of neuralstem cells, culturing the neural stem cells in a hydrogel, wherein thehydrogel is applied to the uniaxially-aligned smooth muscle sheet, andexposing the neural stem cells to at least one extracellular matrix(ECM) component, wherein the ECM component biases differentiation of theneural stem cells into differentiated neural stem cells that areenriched for a neuronal subtype. For example, the neuronal subtype arecholinergic neurons, where the hydrogel can include collagen I, or atleast at least about 800 μg/ml collagen I, or between about 800 μg/mland about 1600 μg/ml collagen I. In another example, the neuronalsubtype are nitrergic neurons, where the hydrogel can include collagenIV and be substantially free of laminin, or at least about 200 μg/mlcollagen IV and be substantially free of laminin. In another example,the neuronal subtype are peptidergic neurons, where the hydrogel caninclude collagen I, collagen IV, and laminin, or at least about 800μg/ml collagen I, at least about 200 μg/ml collagen IV, and at leastabout 5 μg/ml laminin. Another aspect of the invention can includeisolating the differentiated neural stem cells and administering thedifferentiated neural stem cells to a patient. For example, theadministering can include injecting, into the patient, thedifferentiated stem cells in the hydrogel. In another example, thedifferentiated neural stem cells innervate the uniaxially-aligned smoothmuscle sheet to form an innervated smooth muscle sheet and can includethe additional step of implanting the innervated smooth muscle sheetinto a patient.

Another aspect of the invention includes a method of biasing neural stemcell differentiation having the steps of obtaining a population ofneural stem cells, obtaining a population of smooth muscle cells,culturing the neural stem cells in the presence of the smooth musclecells, wherein the neural stem cells adhere on a substrate with asubstrate coating comprising at least one extracellular matrix (ECM)component, wherein the ECM component biases differentiation of theneural stem cells into differentiated neural stem cells that areenriched for neurons. For example, the substrate coating comprises atleast one of laminin, collagen I, and collagen IV, or the substratecoating comprises laminin, and at least one of collagen I and collagenIV, or the substrate coating comprises collagen I and collagen IV, andat least one of laminin and heparan sulfate. Another aspect of theinvention can also include isolating the differentiated neural stemcells and administering the differentiated neural stem cells to apatient. For example, the administering can include injecting thedifferentiated neural stem cells into the patient.

Another aspect of the invention includes a method of biasing neural stemcell differentiation by obtaining a population of neural stem cells,obtaining a population of smooth muscle cells, culturing the neural stemcells in the presence of the smooth muscle cells, wherein the neuralstem cells adhere on a substrate with a substrate coating comprising atleast one extracellular matrix (ECM) component, wherein the ECMcomponent biases differentiation of the neural stem cells intodifferentiated neural stem cells that are enriched for glial cells. Forexample, the substrate coating can include at least collagen I andcollagen IV, and be substantially free of at least one of laminin andheparan sulfate, or the substrate coating can comprises at least 5μg/cm2 collagen I and at least 5 μg/cm2 collagen IV, and besubstantially free of at least one of laminin and heparan sulfate.Another aspect further includes the step of isolating the differentiatedneural stem cells and administering the differentiated neural stem cellsto a patient. For example, the administering comprises injecting thedifferentiated neural stem cells into the patient.

In one aspect of the invention, adult mammalian enteric neuralprogenitor cells can be induced to differentiate on various substratesderived from components or combinations of ECM compositions. Neuronaland glial differentiation was studied as a function of ECM composition.Collagen I and collagen IV substrates supported neuronal differentiationand extensive glial differentiation individually and in combination. Theaddition of laminin or heparan sulfate to collagen substrates improvedneuronal differentiation, increasing the number of neurons and thebranching of neuronal processes and initiation of neuronal networkformation. Various neural ECM components were evaluated individually andin combination to study their effect of neuroglial differentiation ofadult enteric neural progenitor cells.

In another aspect of the invention, tissue-engineered intestinallongitudinal smooth muscle sheets can be innervated using entericneuronal progenitor cells embedded with hydrogels of varying ECMcomposition. Differentiated neuronal composition (cholinergic,nitrergic, peptidergic), as well as functional neuronal physiologymediating smooth muscle contraction/relaxations, were evaluated. Severalfunctional differentiated neuronal subtypes were present intissue-engineered intestinal sheets, capable of mediating smooth musclecontraction/relaxation. Neuronal populations varied from being highlycholinergic (collagen I), highly nitrergic (composite collagen I andcollagen IV), or balanced between the two (composite collagen I andcollagen IV, and laminin and/or heparan sulfate). Additionally, anincrease in peptidergic neurons was detected with laminin and heparansulfate.

The feasibility of transplantation of various types of neuronalprogenitor cells (CNS-derived, neural tube-derived, embryonic and adultENS-derived) in explant cultures of aneural gut is well established.However, conditions required for successful engraftment and long-termsurvival, focusing on a permissive environment heretofore have not beenidentified. Studies related to in vitro differentiation of adult entericneuronal progenitor cells with a focus on the role of the ECM can helpoptimize the survivability and maintenance of both neuronal andneuroglial phenotypes. Moreover, our studies indicate that neuronaldifferentiation can be modulated by varying the composition of ECMmicroenvironments. Enriched populations of differentiated neuronalsubtypes can be derived within transplantable tissue engineered sheets,using ECM microenvironments. ECM microenvironments may also facilitateadequate trophic support and phenotype maintenance of differentiatedneurons.

In certain embodiments, neural differentiation is induced byadministering an effective amount of laminin or a composition comprisinglaminin, including fragments, derivatives, or analogs thereof. In aspecific example, the laminin can be a complete laminin protein. Infurther examples, the laminin is selected from laminin-1, laminin-2,laminin-4, and fragments or combinations thereof. In further examples,the laminin or laminin composition includes a substance at leastsubstantially homologous to laminin-1, laminin-2, or laminin-4. In yetfurther implementations, the laminin or laminin composition comprises apolypeptide at least substantially homologous to the laminin al chain,e.g., having at least 80%, or 85%, or 90%, or 95% sequence identical toat least a fragment of the laminin α1 chain that retains the capacity toinduce neuroglial differentiation.

Amounts effective for various therapeutic treatments of the presentdisclosure may, of course, depend on the severity of the disease and theweight and general state of the subject, as well as the absorption,inactivation, and excretion rates of the therapeutically-active compoundor component, the dosage schedule, and amount administered, as well asother factors known to those of ordinary skill in the art. It alsoshould be apparent to one of ordinary skill in the art that the exactdosage and frequency of administration will depend on the particularlaminin, laminin composition, or other therapeutic substance beingadministered, the particular condition being treated, the severity ofthe condition being treated, the age, weight, general physical conditionof the particular subject, and other medication the subject may betaking. Typically, dosages used in vitro may provide useful guidance inthe amounts useful for in vivo administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of particular disorders. For example, animalmodels of neural disorders may be used to determine effective dosagesthat can then be translated to dosage amount for other subjects, such ashumans, as known in the art. Various considerations in dosagedetermination are described, e.g., in Gilman et al., eds., Goodman AndGilman's: The Pharmacological Bases of Therapeutics, 8th ed., PergamonPress (1990); and Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Co., Easton, Pa. (1990), which is herein incorporated byreference.

In another example, the laminin or laminin composition can be introducedinto an in vitro culture of neural stem cells (or co-administered withneural stem cells to a subject) in an amount sufficient to provide adose of laminin of between about 10 fmol/g and about 500 nmol/g, such asbetween about 2 nmol/g and about 20 nmol/g or between about 2 nmol/g andabout 10 nmol/g. In additional examples, the laminin or laminincomposition can be provided in vitro or administered to a subject in anamount sufficient to provide a dose of laminin of between about 0.01μg/kg and about 1000 mg/kg or between about 0.1 mg/kg and about 1000mg/kg, in particular examples this amount is provided per day or perweek. In another example, the laminin or laminin composition isadministered to a subject in an amount sufficient to provide a dose oflaminin of between about 0.2 mg/kg and about 2 mg/kg. In furtherexamples, the laminin or laminin composition is administered to asubject in an amount sufficient to provide a concentration of laminin inthe administrated material of between about 5 nM and about 500 nM, suchas between about 50 nM and about 200 nm, or about 100 nM.

Addition of heparan sulfate to composite collagen mixtures can improveneuronal differentiation as well. Neuronal networking and neuronalclustering was visible at the later time point. Heparan sulfate and itsinteraction with glial cell-derived neurotrophic factor (GDNF) and otherneurotrophic factors stabilizes and makes these factors locallyavailable, possibly modulating neurite outgrowth and neuronaldifferentiation. Heparan sulfate interacts with both collagen IV andwith laminin, to positive modulate neuronal differentiation, evidencedby the enhanced neurite outgrowth, axonal lengths and initiation ofneuronal networking (FIG. 4 A-H). Composite collagen substrates withlaminin and/or heparan sulfate all maintained a low level of glialfibrillary acidic protein (GFAP) positive glial cells, with initiationof astrocytic networking becoming more obvious at the later time point.In general, substrates that supported neuronal differentiationdemonstrated a bare minimum of glial cells required to possibly supportneuronal cell phenotype or survival.

Taken together, these results help identify optimal 3D matrixcompositions to encapsulate neuronal progenitor cells. In certainembodiments, three dimensional hydrogel environments can also providethe mechanical cues for neural differentiation.

For example, in some embodiments, three dimensional ECM hydrogels cancomprise: collagen I (about 800 μg/ml to about 1600 μg/ml); collagen I(about 800 μg/ml) and collagen IV (about 200 μg/ml); collagen I andcollagen IV with laminin (about 5 μg/ml to about 10 μg/ml); collagen Iand collagen IV with laminin and heparan sulfate (about 10 μg/ml toabout 20 μg/ml); Other components of the gel can include: at least 1%fetal calf serum and at least 0.1× antibiotics in Dulbecco's modifiedEagle's medium. Sodium hydroxide (0.1N) can be used to adjust pH toabout 7.4 for gelation.

Accordingly, methods and systems for treating neurodegenerativeconditions are disclosed whereby neural stem cells (NSCs) can betransplanted into a subject in need such that the cells candifferentiate and ameliorate the neurodegenerative condition. In certainembodiments, the neural stem cells, upon transplantation, generate anamount of neurons or glial cells sufficient to integrate within theneural infrastructure to ameliorate a disease state or condition. In oneembodiment, the disclosed methods include treating neurodegenerativediseases or conditions by transplanting multipotential neuralprogenitors or neural stem cells isolated from the central nervoussystem of a mammal and that have been expanded in vitro and induced todifferentiate by exposure to at least one component of an extracellularmatrix material (ECM).

In another aspect of the invention, treatments can include supplying asuitable number of NSCs to an injured neural area, via transplantation,such that the transplanted cells differentiate into a sufficient numberof neurons and/or glial cells to rehabilitate defective neural circuits.In an embodiment, the disclosed methods include restoring motor functionin a motor neuron disease. A suitable number or a therapeuticallyeffective amount of NSCs or neural progenitors which are capable ofdifferentiating into motor neurons can be provided to at least one areaof neurodegeneration. The NSCs can exert their therapeutic effect byreplacing degenerated neuromuscular junctions.

In certain embodiments, the disclosed methods and systems includeproviding neural stem cells or neural progenitors that integrate withthe host tissue and provide one or more factors to the host neuronsthereby protecting them from degenerative influences present in thetissue. In one preferred embodiment, the disclosed methods includeincreasing differentiation efficiency of transplanted NSCs into neuronsor glial cells by exposure of the stem cells to one or moreextracellular matrix materials (ECMs). The method can also includeexpanding highly enriched NSCs or neural progenitors in theirundifferentiated state and then inducing differentiation so that, upontransplantation, a sufficient number of the cells in the graft adopt adesired phenotype.

The cells of the disclosed methods can be isolated or obtained fromfetal, neonatal, juvenile, adult, or post-mortem tissues of a mammal.The cells of the disclosed methods can be isolated or obtained from thecentral nervous system, blood, or any other suitable source of stemcells that differentiate into neurons. The cells can also be obtainedfrom embryonic stem cells. In certain preferred embodiments, the cellsare autologous neural stem cells obtained from a subject and thatreturned to the subject to ameliorate a neural disorder. In certainembodiments, the neural stem cells can be expanded in culture. In someembodiments, the neural precursor cells can be multipotential NSCscapable of expansion in culture and of generating both neurons and gliaupon differentiation.

The cells can be either undifferentiated, pre-differentiated or fullydifferentiated in vitro at the time of transplantation. In anembodiment, the cells are induced to differentiate into neural lineage.The cells of the disclosed methods can undergo neuronal differentiationin situ in the presence of EMCs and/or pro-inflammatory cytokines andother environmental factors existing in an injured tissue.

Using the present methods, neural circuits can be treated bytransplanting or introducing the cells into appropriate regions foramelioration of the disease, disorder, or condition. Generally,transplantation occurs into nervous tissue or non-neural tissues thatsupport survival of the grafted cells. NSC grafts employed in thedisclosed methods survive well in a neurodegenerative environment wherethe NSCs can exert powerful clinical effects in the form of delaying theonset and progression of neurodegenerative conditions or disease.

The present invention can be used in conjunction with various othertissue engineering methods and compositions including those disclosed incommonly-owned, co-pending Applications No. PCT/US2013/024080 entitled“Innervation of Engineered Structures” filed Jan. 31, 2013 and No.PCT/US2013/024024 entitled “Tubular Bioengineered Muscle Structures”also filed Jan. 31, 2013, each of which is incorporated herein in itsentirety by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A-D are micrographs of rabbitenteric neuro spheres—FIG. 1A is aphase contrast micrograph of rabbit enteric neurospheres in culture.Upon primary isolation and culture, progenitor cells proliferated andaggregated to form neurosphere-like bodies (enteric neurospheres).Immunohistochemistry for initial phenotype (FIGS. 1 B-D): Rabbit entericneurospheres are p75NTR (FIG. 1B), Sox2 (FIG. 1C) and Nestin (FIG. 1D)positive—indicating that they are comprised of neural crest-derivedneuronal and glial progenitor cells. Scale bar 100 um.

FIG. 2 is a schematic illustration of enteric neuro spheredifferentiation as a function of extracellular matrix (ECM) composition:22×11 mm coverslips were coated with poly-L-lysine (PLL), collagen I,collagen IV, laminin or heparan sulfate individually or in combination.Enteric neurospheres were allowed to adhere to the coated coverslips for6 hours. Separately, uncoated glass coverslips were seeded with colonicsmooth muscle cells, and allowed to grow to confluence. In order tostimulate differentiation of enteric neurospheres, a coverslipcontaining confluent smooth muscle was placed within the same dish, sothe two coverslips shared soluble factors.

FIG. 3 illustrates neuronal differentiation on individual coatedcoverslips—βIII Tubulin antibody (white) was used to visualize neuronson day 5 (Figs. A-D) and day 15 (Figs. E-H) coverslips coated with PLL(Figs. A,E), laminin (Figs. B,F), type I collagen (Figs. C,G) and typeIV collagen (Figs. D,H). Enteric neurospheres on PLL barely initiatedneuronal differentiation at day 15. Neurospheres on laminin, collagen Iand collagen IV showed branching and several neuronal processes both atthe early and late time points in vitro.

FIG. 4 illustrates neuronal differentiation on collagen-lamininsubstrates—Neurons stained with βIII tubulin (white) on day 5 (Figs.A-D) and day 15 (Figs. E-H) coverslips coated with type I collagen and 5μm/cm² laminin (Figs. A,E) or 10 μm/cm² laminin (Figs. B,F) or type IVcollagen with 5 (Figs. C,G) or 10 (Figs. D,H) μm/cm² of laminin.Addition of laminin to collagen substrates enhanced early and lateneuronal differentiation, but no significant difference was observablebetween 5 and 10 μg/cm² of laminin. Type IV collagen substrates (Figs.C,D,G,H) demonstrated enhanced neuronal branching and differentiationcompared to type I collagen (Figs. A,B,E,F) substrates.

FIG. 5 illustrates neuronal differentiation on compositecollagen-laminin-heparan sulfate (HS) substrates—Neurons stained withβIII tubulin (white) on day 5 (Figs. A-D) and day 15 (Figs. E-H)coverslips coated with type I and IV collagen with either 5 μm/cm²laminin (Figs. B,F), 0.1 μm/cm² heparan sulfate (HS) (Figs. C,G), both(Figs. D,H) or none (Figs. A,E). Addition of laminin or HS to collagensubstrates enhanced early and late neuronal differentiation, withvisible networking by day 15. Substrates without laminin or HSdemonstrated minimal neuronal differentiation (Figs. A,E).

FIG. 6 illustrates glial differentiation on primary coatedsubstrates—Glia stained with GFAP (white) on day 5 (Figs. A-D) and day15 (Figs. E-H) coverslips coated with poly-L-lysine (PLL) (Figs. A,E),laminin (Figs. B,F), type I collagen (Figs. C,G) and type IV collagen(Figs. D,H). Enteric neurospheres on PLL demonstrated maximal glialdifferentiation starting at day 5 up to day 15. While neurospheres oncollagen substrates demonstrated good early and late glialdifferentiation, laminin coated substrates showed no early glialdifferentiation by day 5 (B), but differentiated subsequently by day 15(F).

FIG. 7 illustrates glial differentiation on collagen-lamininsubstrates—Glia stained with glial fibrillary acidic protein (GFAP)(white) on day 5 (Figs. A-D) and day 15 (Figs. E-H) coverslips coatedwith type I collagen and 5 μm/cm² laminin (Figs. A,E) or 10 μm/cm²laminin (Figs. B,F) or type IV collagen with 5 (Figs. C,G) or 10 (Figs.D,H) μm/cm² of laminin. Addition of laminin to collagen substratespromoted glial differentiation both at days 5 and 15. No significantdifference was observable in GFAP+ glial differentiation between 5 and10 μg/cm² of laminin.

FIG. 8 illustrates glial differentiation on compositecollagen-laminin-heparan sulfate (HS) substrates—Glia stained with GFAP(white) on day 5 (Figs. A-D) and day 15 (Figs. E-H) coverslips coatedwith type I and IV collagen with either 5 μg/cm² laminin (Figs. B,F),0.1 μm/cm² heparan sulfate (Figs. C,G), both (Figs. D,H) or none (Figs.A,E). Addition of laminin or HS to composite collagen substratesdemonstrated glial differentiation that peaked at day 5 (Figs. B-D) thatwas sustained at the later time point (Figs. E-H); and

FIG. 9 illustrates neurite lengths were measured on coated culturesubstrata and compared using one way ANOVA. Two bars for each substrateshow mean neurite lengths at day 5 and day 15. FIG. 9A shows thatneurite lengths on PLL were significantly (***p<0.001) shorter than anyprimary coating substrate. Laminin substrates had the longer neurites(*p<0.05). FIG. 9B shows no significant difference was observed inneurite lengths with the addition of 5 or 10 μg/cm² laminin. FIG. 9Cshows that the addition of laminin or heparan sulphate significantlyincreased neurite lengths over composite collagen substrata (*p<0.05).FIG. 9D shows that mean GFAP immunofluorescence was quantified: PLLsubstrates (***p<0.001) and composite collagen substrates (*p<0.05)supported extensive glial differentiation.

FIG. 10 illustrates scanning electron micrographs of dehydrated ECMgels. Images were obtained at constant magnification and constantworking distance. (A) Collagen I fibers were randomly oriented, andpresented a dense fibrous structure; (B) Composite Collagen I/IV sheetsdemonstrated evidence of the formation of network-like structures; (C)There was no difference in ultrastructure with the addition of laminin;(D) Evidence of cabling and cross-linking was observed with the additionof heparan sulfate. Average porosity was determined and summarized inthe accompanying table. Viscoelastic modulus was calculated usingoscillatory rheometry of ECM gels in their hydrated state, and tabulatedin the table. Scale bar 10 μm.

FIG. 11 illustrates neuronal differentiation within tissue engineeredsheets. Phase contrast micrographs were obtained at the edge of thetissue engineered sheets. Evidence of neuronal differentiation andinitiation of network formation was observed in all ECM compositions atday 10. The arrows indicate instances of preliminary neuronalnetworking. Scale bar 200 μm.

FIG. 12 illustrates immunoblot analysis of tissue engineeredlongitudinal sheets. Sheets were assessed for expression of neuronaldifferentiation, constituent smooth muscle phenotype, and excitatory andinhibitory neural markers. Densitometry was used to quantify bandintensities, to quantify and compare expression. (A) Neuronal βIIITubulin expression was similar amongst all four matrices suggesting thatall ECM compositions supported neuronal differentiation; (B) Constituentsmooth muscle within the tissue engineered sheets maintained contractilephenotype, demonstrated by similar Smoothelin expression; (C) Cholineacetyltransferase (ChAT) expression was significantly (*p<0.05) elevatedin Col I and Col I/IV/Laminin sheets compared to Col I/IV and ColI/IV/Lam/HS sheets; (D) Neuronal nitric oxide synthase (nNOS) expressionwas significantly lower (**p<0.001) in Col I sheets compared to elevatedlevels in all tissue engineered sheets containing Col4 with or withoutlaminin and/or heparan sulfate. (E) Representative immunoblots areprovided along with β Actin, demonstrating equal loading.

FIG. 13 illustrates immunofluorescence for differentiated neurons withintissue engineered sheets. Differentiated neurons within tissueengineered sheets were stained with markers for vasoactive intestinalpeptide (VIP), choline acetyltransferase (ChAT) or neuronal nitric oxidesynthase (nNOS). (A-D) Numerous differentiated VIP-ergic neurons werepresent in tissue engineered sheets. (E-H) Differentiated excitatorycholinergic neurons expressing ChAT were present within tissueengineered sheets; (I-L) Differentiated inhibitory nitrergic neuronsexpressing nNOS were present within tissue engineered sheets. Scale bar100 μm.

FIG. 14 illustrates potassium chloride induced contraction of tissueengineered sheets. 60 mM Potassium chloride (KCl) was used to examinethe electromechanical coupling integrity of the constituent smoothmuscle cells within the tissue engineered sheets. The black tracesindicate the contraction in response to the addition of KCl. The greytraces indicate the addition of KCl in the presence of a neuronalblocker, TTX. Pre-treatment with TTX did not inhibit KCl-inducedcontraction. The ECM composition of the tissue engineered sheets did notaffect smooth muscle contraction, evidenced by similar contractilepatterns in response to KCl stimulation. A robust and immediatecontraction was observed upon addition of KCl (indicated by the arrows)in all tissue engineered sheets (A-D), similar to native rabbitintestinal tissue (E). Peak contraction in response to KCl rangedbetween 279.5 μN and 296.5 μN in tissue engineered sheets, and averagedat 373.3±10.63 in native tissue. (F) The area under the curve ofKCl-induced contraction was quantified to demonstrate no significant(ns) difference in contraction in tissue engineered sheets, with aslightly elevated (*p<0.05) magnitude in native tissue.

FIG. 15 illustrates acetylcholine induced contraction. Addition of 1 μMAcetylcholine (Ach; arrow) resulted in contraction of tissue engineeredsheets, as well as native tissue. Gray traces demonstrate Ach treatmentin the presence of neuronal blocker, TTX. (A-E) Representative tracingsof Ach-induced contraction in tissue engineered sheets and nativetissue. Magnitude of Ach-induced contraction varied between tissueengineered sheets. Comparison of the area under the curve of contractiondemonstrated that tissue engineered sheets approached 31.5% (Col4)-67.6%(Laminin) of contraction observed in native tissue. In the presence ofTTX, magnitude of Ach-induced contraction was attenuated. Quantificationof inhibition (F) revealed that the degree of inhibition with TTX variedamongst the tissue engineered sheets with different ECM compositions.Highest inhibition was observed in Col i (72.77±2.5%) and ColI/IV/Laminin (60.58±1.7%) sheets, indicating an elevated presence ofcholinergic neurons contributing to Ach-induced contraction.Significantly lower inhibition (*p<0.05; 48.36±4.3-50.31±4.2%) was seenin Col I/IV and Col I/IV/Lam/Heparan sulfate sheets. TTX pre-treatmentattenuated Ach-induced contraction by 72.73±3.7% in native tissue.

FIG. 16 illustrates electrical Field Stimulation induced relaxation intissue engineered sheets. Electrical Field stimulation (EFS; shaded grayarea) was used to stimulate relaxation in tissue engineered sheets (A-D)and native tissue (E). Grey traces indicate TTX pre-treatment. EFSinduced relaxation was significantly attenuated by TTX-pretreatment(90.9±2.41%-94.41±0.93%), indicating that differentiated neurons withinthe tissue engineered sheets were capable of evoking smooth musclerelaxation. The magnitude of relaxation varied amongst the tissueengineered sheets (summarized in Table 3). (F) Quantification of thearea under the curve of relaxation indicated that Col I sheets had asignificantly low (***p<0.001; 23142±4921 AU) magnitude of relaxation.Relaxation in Col I/IV sheets (109693±8465 AU) were similar to thoseobserved in native tissue (101550±11279 AU) in response to theelectrical field, indicating the presence of elevated levels ofinhibitory motor neurons capable of mediating relaxation. Relaxation washigher in Col I/IV/Laminin (69025±7154 AU) and Col I/IV/Lam/Heparansulfate (68395±8228 AU) sheets, when compared to Col I, also indicatinga similar increase in the presence of inhibitory motor neurons.

FIG. 17 illustrates inhibition of relaxation with L-NAME. Thefunctionality of nitrergic neurons was studied by inhibiting EFS-inducedrelaxation with L-NAME, a non-metabolizable substrate for nNOS. The greytraces indicate EFS in the presence of L-NAME. Pretreatment with L-NAMEattenuated EFS-induced relaxation in all tissue engineered sheets (A-D)and native tissue (E). (F) Quantification of the area under the curvefor relaxation indicated that the extent of L-NAME inhibition variedamongst the tissue engineered sheets. Col I sheets had a significantlylower % inhibition with L-NAME (*p<0.05; 33.4±8.4%) corresponding to thelowest immunoblot expression of nNOS. The degree of L-NAME inhibitionwas higher in tissue engineered sheets containing Col I/IV and/orlaminin and/or heparan sulfate (57.16%-62.28%), corresponding to thehigher immunoblot expression of nNOS. Attenuation of relaxation in thepresence of L-NAME was 78±2.9% in native tissue.

FIG. 18 illustrates inhibition of relaxation with VIP-Ra. Thefunctionality of VIP-ergic neurons was studied by inhibiting EFS-inducedrelaxation with VIP-Ra, a VIP-receptor antagonist. Grey traces indicateEFS in the presence of VIP-Ra. In the presence of VIP-Ra, EFS-inducedrelaxation was attenuated in tissue engineered sheets (A-D) and innative tissue (E), indicating the presence of functional VIP-ergicneurons capable of mediating smooth muscle relaxation upon electricalfield stimulation. Area under the curve of relaxation was quantified, tocalculate the % inhibition of relaxation in the presence of VIP-Ra (F).Extent of VIP-Ra induced inhibition of relaxation varied from56.55±3.12%-63.11±3.2% in tissue engineered sheets, and averaged at73.32±3.23% in native tissue.

DETAILED DESCRIPTION Definitions

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. The steps of any method canbe practice in feasible order and are restricted to a sequential ordermerely because they are so recited in a claim.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

“Differentiation” refers to a change that occurs in cells to cause thosecells to assume certain specialized functions and to lose the ability tochange into certain other specialized functional units. Cells capable ofdifferentiation may be any of totipotent, pluripotent or multipotentcells. Differentiation may be partial or complete with respect to matureadult cells.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts. Neural stem cellscan be isolated from embryonic and adult central nervous system (CNS)tissue, neural tube tissue or enteric nervous system (ENS) tissue.

Stem cells can be further classified according to their developmentalpotential as: (1) totipotent; (2) pluripotent; (3) multipotent; (4)oligopotent; and (5) unipotent. Totipotent cells are able to give riseto all embryonic and extra-embryonic cell types. Pluripotent cells areable to give rise to all embryonic cell types. Multipotent cells includethose able to give rise to a subset of cell lineages, but all within aparticular tissue, organ, or physiological system (for example,hematopoietic stem cells (HSC) can produce progeny that include HSC(self-renewal), blood cell-restricted oligopotent progenitors, and allcell types and elements (e.g., platelets) that are normal components ofthe blood). Cells that are oligopotent can give rise to a morerestricted subset of cell lineages than multipotent stem cells; andcells that are unipotent typically are only able to give rise to asingle cell lineage.

In a broader sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase “differentiates into a neural lineage orphenotype” refers to a cell that becomes partially or fully committed toa specific neural phenotype of the CNS or PNS, i.e., a neuron or a glialcell, the latter category including without limitation astrocytes,oligodendrocytes, Schwann cells and microglia. The term “neural” as usedherein is intended to encompass all electrical active cells, e.g., cellsthat can process or transmit information through electrical or chemicalsignals, including the aforementioned neurons, glial cells, astrocytes,oligodendrocytes, Schwann cells and microglia.

For the purposes of this disclosure, the terms “neural progenitor cell”or “neural precursor cell” mean a cell that can generate progeny thatare either neuronal cells (such as neuronal precursors or matureneurons) or glial cells (such as glial precursors, mature astrocytes, ormature oligodendrocytes). Typically, the cells express some of thephenotypic markers that are characteristic of the neural lineage.Typically, they do not produce progeny of other embryonic germ layerswhen cultured by themselves in vitro, unless dedifferentiated orreprogrammed in some fashion.

A “neuronal progenitor cell” or “neuronal precursor cell” is a cell thatcan generate progeny that are mature neurons. These cells may or may notalso have the capability to generate glial cells. A “glial progenitorcell” or “glial precursor cell” is a cell that can generate progeny thatare mature astrocytes or mature oligodendrocytes. These cells may or maynot also have the capability to generate neuronal cells.

The phrase “biocompatible substance” and the terms “biomaterial” and“substrate” are used interchangeably and refer to a material that issuitable for implantation or injection into a subject. A biocompatiblesubstance does not cause toxic or injurious effects once implanted inthe subject. In one embodiment, the biocompatible substrate includes atleast one component of extracellular matrix. In other embodiments, thesubstrate can also include a polymer with a surface that can be shapedinto the desired structure that requires repairing or replacing. Thepolymer can also be shaped into a part of a body structure that requiresrepairing or replacing. In another embodiment, the biocompatiblesubstrate can be injected into a subject at a target site.

In one embodiment, the substrate is an injectable or implantablebiomaterial that can be composed of crosslinked polymer networks whichare typically insoluble or poorly soluble in water, but can swell to anequilibrium size in the presence of excess water. For example, ahydrogel can be injected into desired locations within the organ. In oneembodiment, the collagen can be injected alone. In another embodiment,the collagen can be injected with other hydrogels. The hydrogelcompositions can include, without limitation, for example, poly(esters),poly(hydroxy acids), poly(lactones), poly(amides), poly(ester-amides),poly(amino acids), poly(anhydrides), poly(ortho-esters),poly(carbonates), poly(phosphazines), poly(thioesters), polysaccharidesand mixtures thereof. Furthermore, the compositions can also include,for example, a poly(hydroxy) acid including poly(alpha-hydroxy) acidsand poly(beta-hydroxy) acids. Such poly(hydroxy) acids include, forexample, polylactic acid, polyglycolic acid, polycaproic acid,polybutyric acid, polyvaleric acid, and copolymers and mixtures thereof.

Hydrogels with effective pore sizes in the 10-100 nm range and in the100 nm-10 micrometer range are termed “microporous” and “macroporous”hydrogels, respectively. Microporous and macroporous hydrogels are oftencalled polymer “sponges.” When a monomer, e.g., hydroxyethylmethacrylate (HEMA), is polymerized at an initial monomer concentrationof 45 (w/w) % or higher in water, a hydrogel is produced with a porosityhigher than the homogeneous hydrogels. Hydrogels can also expand in thepresence of diluent (usually water). The matrix materials of presentinvention encompass both conventional foam or sponge materials and theso-called “hydrogel sponges.” For a further description of hydrogels,see U.S. Pat. No. 5,451,613 (issued to Smith et al.) herein incorporatedby reference.

The term “extracellular matrix” or “ECM” is used herein to denotecompositions comprising one or more of the following: collagen I,collagen IV, laminin, heparan sulfate, or fragments of one or more ofsuch proteins.

“Collagen I” refers to collagen I or collagen I compositions derivedfrom cell culture, animal tissue, or recombinant means, and may bederived from human, murine, porcine, or bovine sources. “Collagen I”also refers to substances or polypeptide(s) at least substantiallyhomologous to collagen I or collagen I compositions. Additionally,“collagen I” refers to collagen I or collagen I compositions that do notinclude a collagen I fragment, e.g., including essentially only acomplete collagen I protein.

“Collagen IV” refers to collagen IV or collagen IV compositions derivedfrom cell culture, animal tissue, or recombinant means, and may bederived from human, murine, porcine, or bovine sources. “Collagen IV”also refers to substances or polypeptide(s) at least substantiallyhomologous to collagen IV or collagen IV compositions. Additionally,“collagen IV” refers to collagen IV or collagen IV compositions that donot include a collagen IV fragment, e.g., including essentially only acomplete collagen I protein.

“Laminin” refers to laminin, laminin fragments, laminin derivatives,laminin analogs, or laminin compositions derived from cell culture,recombinant means, or animal tissue. “Laminin” can be derived fromhuman, murine, porcine, or bovine sources. “Laminin” refers to lamininor laminin compositions comprising laminin-1, laminin-2, laminin-4, orcombinations thereof. “Laminin” also refers to substances orpolypeptide(s) at least substantially homologous to laminin-1,laminin-2, or laminin-4. Additionally, “laminin” refers to laminin orlaminin compositions that do not include a laminin fragment, e.g.,including essentially only a complete laminin protein.

The term “substantially free of laminin” and “free of laminin” are usedinterchangeably herein to denote compositions in which laminin is absentor present in such low concentrations that it does not play anysignificant role in neural stem cell differentiation, e.g., wherelaminin is only present in concentrations less than 5 μg/ml in hydrogelsor 5 μg/cm on substrate coatings, or more preferably less than 2 μg/mlin hydrogels or 2 μg/cm² on substrate coatings, or less than 1 μg/ml inhydrogels or 1 μg/cm on substrate coatings, and in some instances lessthan 0.1 μm/ml in hydrogels or 0.1 μm/cm² on substrate coatings.

The term “substantially free of heparan sulfate” and “free of heparansulfate” are used interchangeably herein to denote compositions in whichheparin sulfate is absent or present in such low concentrations that itdoes not play any significant role in neural stem cell differentiation,e.g., where heparan sulfate is only present in concentrations less than5 μg/ml in hydrogels or 5 μg/cm² on substrate coatings, or morepreferably less than 2 μg/ml in hydrogels or 2 μg/cm² on substratecoatings, or less than 1 μg/ml in hydrogels or 1 μg/cm² on substratecoatings, and in some instances less than 0.1 μm/ml in hydrogels or 0.1μm/cm² on substrate coatings.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. Included areTeratocarcinomas and embryonic stem cells: A practical approach (E. J.Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in MouseDevelopment (P. M. Wasserman et al. eds., Academic Press 1993);Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth.Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells:Prospects for Application to Human Biology and Gene Therapy (P. D.Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998),

For elaboration of nervous system abnormalities, and thecharacterization of various types of nerve cells, markers, and relatedsoluble factors, the reader is referred to CNS Regeneration: BasicScience and Clinical Advances, M. H. Tuszynski & J. H. Kordower, eds.,Academic Press, 1999.

Methods in molecular genetics and genetic engineering are described inMolecular Cloning: A Laboratory Manual, 2nd Ed. (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture(R. I. Freshney, ed., 1987); the series Methods in Enzymology (AcademicPress); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P.Calos, eds., 1987); Current Protocols in Molecular Biology and ShortProtocols in Molecular Biology, 3rd Edition (F. M. Ausubel et al., eds.,1987 & 1995); and Recombinant DNA Methodology II (R. Wu ed., AcademicPress 1995). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, and ClonTech.

General techniques used in raising, purifying and modifying antibodies,and the design and execution of immunoassays includingimmunohistochemistry are described in Handbook of ExperimentalImmunology (D. M. Weir & C. C. Blackwell, eds.); Current Protocols inImmunology (J. E. Coligan et al., eds., 1991); and R. Masseyeff, W. H.Albert, and N. A. Staines, eds. Methods of Immunological Analysis(Weinheim: VCH Verlags GmbH, 1993).

Sources of Stem Cells

This invention can be practiced using stem cells of various types, whichmay include the following non-limiting examples: U.S. Pat. No. 5,851,832reports multipotent neural stem cells obtained from brain tissue. U.S.Pat. No. 5,766,948 reports producing neuroblasts from newborn cerebralhemispheres. U.S. Pat. Nos. 5,654,183 and 5,849,553 report the use ofmammalian neural crest stem cells. U.S. Pat. No. 6,040,180 reports invitro generation of differentiated neurons from cultures of mammalianmultipotential CNS stem cells. WO 98/50526 and WO 99/01159 reportgeneration and isolation of neuroepithelial stem cells,oligodendrocyte-astrocyte precursors, and lineage-restricted neuronalprecursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtainedfrom embryonic forebrain and cultured with a medium comprising glucose,transferrin, insulin, selenium, progesterone, and several other growthfactors.

Except where otherwise required, the invention can be practiced usingstem cells of any vertebrate species. Included are stem cells fromhumans; as well as non-human primates, domestic animals, livestock, andother non-human mammals.

Neural Glial Differentiation

Enteric neuronal progenitor cells have been identified in the adultmammalian gut, and have been isolated from humans up to and over 80years of age. Previously, several groups have shown that a self-renewingpopulation of Sox 2, Sox10, Nestin and p75 positive neural-crest derivedprogenitor cells can be isolated either from full-thickness, muscularisor mucosal biopsies of the adult mammalian gut. These cells have beendemonstrated to have the potential to differentiate into severalneuronal subtypes including inhibitory and excitatory motor neurons andglia.

Various types of neuronal progenitor cells (CNS-derived, neuraltube-derived, embryonic and adult ENS-derived) from explant cultures ofaneural gut can also be transplanted. Alterations in the extracellularmatrix of the gut mesenchyme has been documented in aganglionic regionsof rodent gut, suggesting the importance of a permissive extracellularenvironment to promote effective in utero colonization anddifferentiation of neural crest cells in the developing gut. Sincetransplantation and subsequent functional neo-innervation is oneclinical goal of neural stem cell transplantation, in vitro studiesshould mimic developmental conditions in vivo, in terms of providing apermissive and favorable ECM (such as a three-dimensional environment).Understanding the role of the ECM in affecting neuroglialdifferentiation of adult enteric neuronal progenitor cells can enhancethe survivability and maintenance of a stable phenotype upontransplantation.

Mammalian myenteric ganglia in vivo are surrounded by a matrix comprisedpredominantly of type IV collagen, laminin, heparan sulphateproteoglycan, and entactin. The enteric plexus lacks large connectivetissue spaces for blood vessels like the peripheral nervous system. Thetwo-dimensional culture substratum may modulate neuronal and glialdifferentiation based on ECM composition. Different ECM components mayinfluence enteric glia and neurons come in to contact with in vivo inthe adult myenteric plexus, such as collagen IV, laminin and heparansulfate.

Addition of laminin to collagen substrates unexpectedly improved neuriteoutgrowth with longer neurite lengths (compare 156.1±7.2 μm to 215.1±7.6μm). while there was an overall enhancement in neuronal differentiationas well as neurite outgrowth, there was no significant differencebetween the additions of 5 or 10 μg/cm² of laminin. This empiricaldetermination was important in determining a minimal amount of lamininthat can influence neuroglial differentiation without affecting neuriteoutgrowth adversely in a situation that requires neo-innervation ofdenervated tissues.

Addition of heparan sulfate to composite collagen mixtures improvedneuronal differentiation as well. Neuronal networking and neuronalclustering was visible at the later time point. Heparan sulfate mayinteract with GDNF and other neurotrophic factors to stabilize and makethe factors locally available. Heparan sulfate interacts with bothcollagen IV and with laminin, to positively modulate neuronaldifferentiation. In one embodiment, heparan sulfate is added to thecollagen mixture.

Composite collagen substrates with laminin and/or heparan sulfate allmaintained a low level of GFAP positive glial cells, with initiation ofastrocytic networking becoming more obvious at the later time point. Ingeneral, substrates that supported neuronal differentiation demonstrateda bare minimum of glial cells required to possibly support neuronal cellphenotype or survival.

Substrates that supported neuronal differentiation may result inenriched populations of neuronal cells comprising greater than 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, or anyintermediate percentage.

Enteric neurospheres demonstrated a tendency to differentiate into gliaon PLL coated substrates as well as on composite collagen substrates inthe absence of laminin and heparan sulfate. In contract, culturesubstrates with laminin and heparan sulfate promoted extensive neuronaldifferentiation while simultaneously supporting only a minimal glialcell population. Laminin and collagen IV coated coverslips positivelymodulated neuronal differentiation by increased number of neurites perneuron and longer neurite lengths compared to fibrillar collagen I (FIG.2B-D, F-H).

Taken together, these results identify suitable 3D matrix compositionsto deliver neuronal progenitor cells. Three dimensional hydrogelenvironments also provide the mechanical cues for neuraldifferentiation, more readily translatable to in vivo conditions thaninfinitely stiff glass substrates.

The extracellular matrix (ECM) plays an enormous role in dictating stemcell fate. The ECM composition, structure and mechanical properties canall modulate progenitor cell differentiation. The adult mammalianmyenteric ganglia are surrounded by an extracellular matrix primarilycomposed of collagen IV, laminin and a heparan sulfate proteoglycan,with enteric glia always in direct contact with the ECM. Enteric neuronsalso come in direct contact with this ECM, though much less frequentlythan glia. Laminin, fibronectin and proteoglycans are expressed withinthe embyonic gut to aid its colonization by vagal neural crest cells.Collagen IV is distributed in the developing nervous system along theneural crest. Additionally, laminin promotes neural cell adhesion andaxonal outgrowth. Heparan sulfate is important for GDNF signaling in thegut, and stabilizes and influences neuronal differentiation in vitro.

It has been discovered that components of neural ECM can affect thedifferentiation of gut-derived neuronal progenitor cells of neural-crestlineage. Two timepoints were defined to identify early and latedifferentiation events—day 5 (early) and day 15 (late) based on previousexperiments. Immunohistochemistry for βIII tubulin (neuron specificmicrotubule) and GFAP (Glial fibrillary acidic protein) was used toidentify differentiated neurons and glia on coated culture substrata.

Substrates that supported glial differentiation may result in enrichedpopulations of glial cells comprising greater than 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, or anyintermediate percentage.

Neuronal Subtype Differentiation

Neural stem cell transplantation is a promising therapeutic approach torepopulate neurons within enteric ganglia. A complete loss of neurons isreported in HSCR, and a partial loss of selective neuronal subtypes isdocumented in achalasia and stenosis. Several groups have injectedenteric neuronal progenitor cells into experimental models ofaganglionosis, demonstrating the feasibility of transplantation.However, there is inadequate focus on differentiation of progenitorcells into mature neuronal subtypes, and subsequent assessment offunctionality. Here, we describe one embodiment of the invention,whereby we describe a novel method to bias differentiation of entericneuronal progenitor cells in vitro, prior to transplantation.

The ECM microenvironment, consisting of collagens, laminin andproteoglycans, not only acts as a structural framework for cells, butalso plays an active role in aiding neurotrophic signaling. In anembodiment of this invention, four ECM components (collagen I, collagenIV, laminin and heparan sulfate) were evaluated, three of which areknown to be present in adult myenteric ganglia. Collagen IV has beendocumented to be favorable for neurite outgrowth and neuronaldifferentiation. Laminin has long been known for its neurite promotingactivity, in central, peripheral, and enteric neurons. The role of theheparan sulfate proteoglycan in neuronal differentiation is also welldocumented, both developmentally and in regenerative medicineapplications. Fibrillar Collagen I was used additionally in thesestudies for ease of gelation and incorporation of other ECM componentswithin a 3D hydrogel.

Apart from composition, substrate elasticity has been demonstrated toaffect the differentiation of adult neural stem cells, with neuronaldifferentiation reported between 100-500 Pa. ECM hydrogel compositionswere adjusted in order to maintain their viscoelastic modulus within therange suitable for neuronal differentiation (FIG. 10, table). Structuralarchitecture was verified using scanning electron microscopy, whereinthe addition of collagen IV demonstrated the presence of networkstructures, similar to self-assembled collagen IV in the mammalianbasement membrane. The addition of laminin did not alter theultrastructure, because it was expected to coat collagen fibers evenly.Additionally, laminin was also not expected to alter thestiffness/viscoelasticity of the gels, given the manner of itsinteraction with the collagen. The glycosaminoglycan chains of heparansulfate are documented to cross link between laminin and collagen IV,thereby pulling fibers into a more compact structure, and slightlyincreasing the viscoelasticity of ECM gels.

Smooth muscle cells within tissue engineered sheets drive thedifferentiation of enteric neuronal progenitor cells. Tissue engineeredsheets provided a good modality to assess variability of differentiatedneurons due to ECM composition as well as the functionality ofdifferentiated neurons. The proximity to smooth muscle promoted thedifferentiation of enteric neuronal progenitor cells extensively. Invitro differentiation of neural stem cells in the presence ofgut-derived factors has been demonstrated previously by us and others.Neurotrophic factors (NT-3, Neurturin, GDNF) and morphogens (BMP-2/4)capable of driving enteric neuronal progenitor cell proliferation anddifferentiation have been demonstrated to arise from the smooth muscleand mesenchyme of the developing and adult gut. Recently, the postnatalbowel was demonstrated to support the differentiation of entericneuronal progenitor cells, strengthening the fact that cues fordifferentiation can be derived from the postnatal gut. Hence, it wasexpected that smooth muscle cells within the tissue engineered sheetswould drive the differentiation of enteric neuronal progenitor cells. Weevaluated all tissue engineered sheets to ensure that the constituentsmooth muscle cells demonstrated a contractile phenotype expressingSmoothelin (FIG. 14B). Smoothelin expression has been previouslydemonstrated to be essential for contractility of smooth muscle. In linewith the equivalent expression of smoothelin, myogenic electromechanicalcoupling integrity was also equivalent in the tissue engineered sheets(FIG. 14A-D) Similar patterns of contractions were observed in tissueengineered sheets in response to KCl, regardless of ECM composition.

The ECM modulates differential neuronal subtypes while supportingoverall smooth muscle-driven neuronal differentiation. In the presenceof the smooth muscle, enteric neuronal progenitor cells differentiated,and expressed similar amounts of pan-neuronal marker βIII Tubulin (FIG.14A), suggesting that smooth muscle derived factors and substrateviscoelasticity were suitable for overall neuronal differentiation.However, on closer examination of neural subtypes, there was adifferential expression of excitatory and inhibitory markers withintissue engineered sheets with varying ECM compositions (FIG. 14). Sheetscontaining laminin had a balanced expression of both ChAT and nNOS.Kinetics of Ach-induced contraction in laminin sheets was most similarto native tissue, indicating the presence of an increased viablecholinergic neuronal component in composite collagen/laminin sheets.Furthermore, attenuation of EFS-induced relaxation by L-NAME (˜62%) wasalso kinetically similar to native tissue (˜78%), indicating thepresence of a nitrergic neuronal component.

Collagen I, in the absence of any other matrix components, was the ECMof choice when an enriched cholinergic neuronal population was required,with a significantly diminished nitrergic neuronal population (FIG.14C-D). While these sheets demonstrated a robust TTX-sensitiveAch-induced contraction commensurate with the heightened ChAT proteinexpression, relaxation in response to an electrical field wasdiminished. Furthermore, there was minimal attenuation of relaxationupon the inhibition of nNOS, correlating with the low nNOS expression.

Substrates that supported cholinergic neuron differentiation may resultin enriched populations of cholinergic neurons comprising greater than25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,100%, or any intermediate percentage.

Composite Collagen I/IV sheets had an enhanced nNOS protein expression,with an associated increase in EFS induced relaxation (FIG. 18B). AUC ofrelaxation in composite Collagen I/IV sheets was comparable to nativeintestinal tissue. However, both ChAT expression and contraction waslower in composite collagen sheets.

Substrates that supported nitrergic neuron differentiation may result inenriched populations of nitrergic neurons comprising greater than 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%,or any intermediate percentage.

The ECM is a framework upon which smooth muscle derived factors regulatedifferentiation of neural subtypes. We demonstrate a critical role ofcollagen I and collagen IV containing ECM environments in promotingexcitatory and inhibitory motor neurons, respectively. The ECMmicroenvironment plays a role in modulating neurotrophic as well asmorphogenetic signaling. Morphogenetic signaling via the BMP familyexpressed in fetal gut is important for the phenotypic diversity ofenteric ganglia, including nitrergic and VIP-ergic neurondifferentiation. Collagen IV is documented to modulate BMP signaling,and heparan sulfate modulates GDNF signaling in the gut.Immunoreactivity of neurotrophic factor, NT-3, has been observed inganglia and in the ECM molecules surrounding them, suggesting a role fora Collagen IV-based ECM to modulate NT-3 signaling. The constituentsmooth muscle phenotype in tissue engineered sheets was contractile,expressing smoothelin, and generating contractions and relaxationsapproaching ˜60% of those generated by native intestinal tissue.Differentiation cues arising from the constituent smooth muscle cellsdrove enteric neuronal differentiation. Furthermore, it is likely thatthe ECM could act as a framework for smooth muscle-derived factors,enhancing or inhibiting their effects, resulting in the generation ofdifferential neuronal phenotypes.

EXAMPLES Reagents

All tissue culture reagents were purchased from Invitrogen (Carlsbad,Calif.) unless specified otherwise. Primary and fluorophore conjugatedsecondary antibodies were purchased from Abcam (Cambridge, Mass.). Rattail type I collagen and natural mouse type IV collagen were purchasedfrom BD Biosciences (Bedford, Mass.) and laminin was from Invitrogen(Carlsbad, Calif.). Heparan sulfate was purchased from Celsus Labs(Cincinnati, Ohio).

Isolation of Rabbit Enteric Neuronal Progenitor Cells and IntestinalSmooth Muscle Cells.

New Zealand white rabbits were euthanized using ketamine/xylazine.Smooth muscle cells were isolated and cultured using standard protocols(See, for example, Somara et al. Am J Physiol Gastrointest LiverPhysiol. 2006; 291(4):G630-9). For the isolation of enteric neuronalprogenitor cells, 5 cm² biopsies were dissected from the jejunum, andretrieved in Hank's Buffered Salt Solution (HBSS) with 2×antibiotics/antimycotics and 1× gentamicin sulfate. Luminal content wascleaned and tissues were washed extensively with HBSS. Enteric neuronalprogenitor cells were isolated from these tissues using acollagenase/dispase digestion method. (see, for example, Almond et al.,Characterisation and transplantation of enteric nervous systemprogenitor cells. Gut. 2007; 56(4):489-96. PMCID: 1856871). Cells wereplated on to bacterial petri dishes in neuronal growth media(Neurobasal+1× N2 supplement+1× antibiotics) following filtrationthrough a 40 μm mesh.

Isolation and Culture of Rabbit Longitudinal Smooth Muscle Cells (LSMCs)

Rabbit sigmoid colon was removed by dissection, and relieved of fecalcontent. The tissue was kept on ice and moist with Hank's balanced saltsolution (HBSS) containing antibiotics and sodium bicarbonate. Thecleaned colon was slipped onto a plastic pipette. Blood vessels andadherent fat were picked off with forceps. Kimwipe® (Kimberly-Clark,Neenah, Wis.) wetted with HBSS was used to wipe the outer layer of thecolon. Fine-tip forceps were used to pick off the longitudinal musclelayer from the colon and store them in ice-cold HBSS. The tissue wasfinely minced, digested twice with type II collagenase (0.1%) at 32° C.for 1 h, and filtered through a 500-μm Nytex® (Tetko, Elmsford, N.Y.)mesh. The filtrate was washed three times and plated in DMEM with 10%FBS, 1.5% antibiotics, and 0.5% L-glutamine onto regular tissue cultureflasks.

Immunohistochemical Characterization of Rabbit Enteric Neurospheres

In order to characterize the initial phenotype of rabbit entericneurospheres in culture, neurospheres were harvested by centrifugationat 1000 g for 10 minutes in microfuge tubes. The growth media was gentlyaspirated, and neurospheres were fixed with 3.7% neutral bufferedformaldehyde and blocked with 10% horse serum. Primary antibodies forp75 (Millipore, Billerica Mass.), Sox2 and Nestin were incubated for 30minutes at room temperature. Unbound antibody was washed using phosphatebuffered saline (PBS), and appropriate fluorophore-conjugated secondaryantibodies were incubated for an additional 30 minutes. Neurosphereswere mounted using Prolong Gold antifade mounting medium (Invitrogen,Carlsbad Calif.), and visualized using an inverted Nikon TiE fluorescentmicroscope.

Rheological Characterization of ECM Hydrogels

Oscillatory rheometry (ATS RheoSystems) was used to measure viscoelasticmoduli of ECM gels. 20 mm parallel base plates were used to perform astress sweep of the sample at 1 Hz. ECM gels were allowed to gel in situbetween the parallel plates at 37° C. The viscoelastic modulus wasobtained from a linear region of the stress-strain curve, at strainslower than 10%, within the sensitivity ranges for torque and strain ofthe rheometer. 3-5 individually manufactured ECM gels were measured todetermine an average viscoelastic modulus. Compositions that resulted ina matrix viscoelasticity within the range of 150-300 Pa were utilizedfor further experimentation, so as not to let stiffness be a variable ininfluencing neuroglial differentiation.

Characterization of Ultrastructure of ECM Hydrogels

Sample preparation of ECM hydrogels for scanning electron microscopy wasadapted from Stuart et al. [31]. Gels were dehydrated through gradedethanol (10% to 100%). Hydrogels were dried at critical point usingcarbon dioxide exchange. The resulting dehydrated ECM discs were mountedonto metallic stubs with conducting carbon tape, sputter coated withgold, and visualized using an AMRAY 1910 Field Emission ScanningElectron Microscope. Constant working distance and magnification weremaintained to image all samples. NIH Image J was used to measure andcompare fiber diameters. Porosity was determined using Image J frommicrographs obtained from at least three-independent samples ofdehydrated ECM gels.

Tissue Engineering Innervated Intestinal Smooth Muscle Sheets

Briefly, 500,000 longitudinal smooth muscle cells were aligneduniaxially for 4 days on 35 mm diameter circular Sylgard moldscontaining wavy microtopographies. Enteric neurospheres were treatedwith Accutase to obtain single cell suspensions. 200,000 cells wereresuspended in the appropriate ECM solution and overlaid on the alignedsmooth muscle monolayer. Upon gelation, neuronal differentiation medium(neurobasal-A) was added, supplemented with B27 and 1% fetal bovineserum. Differentiation medium was exchanged every second day. Entericneuronal progenitor cells were allowed to differentiate within thehydrogel for a period of 10 days. Smooth muscle cells compacted the ECMhydrogel over the next 10 days, forming ˜1 cm long innervated smoothmuscle sheets, anchored between silk sutures. Phase microscopy was usedto image neuronal differentiation at the edge of the tissue engineeredsheets.

Biochemical Characterization of Neuroglial Composition in TissueEngineered Sheets

At day 10, tissue engineered sheets were harvested inradioimmunoprecipitation buffer to isolate protein. Proteinconcentration was estimated spectrophotometrically using the Bradfordassay. 20 μg of protein from each sample was resolvedelectrophoretically and transferred to polyvinylidene difluoridemembranes. Membranes were blotted with antibodies for neuronal βIIITubulin, neuronal nitric oxide synthase (nNOS), cholineacetyltransferase (ChAT), and Smoothelin. β-Actin was used to confirmequal loading. HRP-conjugated secondary antibodies were used tovisualize proteins using enhanced chemiluminescence.

Immunohistochemical Characterization of Neuron Composition in TissueEngineered Sheets

Tissue engineered sheets were fixed in 4% formaldehyde and washedextensively in glycine buffer. Immunohistochemical staining wasperformed following previously established protocols utilized forstaining differentiated neurons within bioengineered tissues. Sheetswere blocked with 10% horse serum and permeabilized in 0.15% Triton-Xfor 45 minutes. Permeabilized sheets were incubated with primaryantibodies directed against Vasoactive Intestinal Peptide (VIP), ChATand nNOS for 60 minutes at room temperature. Following antibodyincubation, sheets were washed three times with phosphate bufferedsaline, pH 7.4. Tissue engineered sheets were incubated with appropriatefluorophore conjugated secondary antibodies for 45 minutes, washed inphosphate buffered saline and imaged using an inverted fluorescencemicroscopy (Nikon Ti-E, Japan). For a negative control, incubation withthe primary antibody was skipped, and only fluorophore conjugatedsecondary antibodies were used to visualize background fluorescence.

Measurement of Physiological Function in Inntervated Tissue EngineeredSheets

Myogenic and neuronal functionality were assessed using real-time forcegeneration as previously described [30, 33]. 4-5 individual tissueengineered sheets for each ECM composition were tested. Tissueengineered sheets were anchored between a stationary pin and measuringpin of a force transducer (Harvard Apparatus, Holliston Mass.) at 0%stretch. The organ bath maintained temperature at 37° C. An additional10% stretch was applied using a vernier control. Tissues were immersedin 4 ml of medium, which was exchanged at the end of every experimentfollowing a brief wash with fresh medium. Peak contraction or maximalrelaxation was quantified following pharmacological or electricalstimuli, and compared between tissue engineered sheets with varying ECMcompositions. Before each treatment, tissues were washed in fresh warmmedium and allowed to equilibrate to a baseline. The following stimuliwere used independently to assess physiological functionality of thetissue engineered sheets: 1) 60 mM Potassium chloride to assesselectromechanical coupling integrity of the smooth muscle; 2) 1 μMAcetylcholine (contractile agonist); 3) Electrical field stimulation (5Hz, 0.5 ms, 40V) applied using parallel plate platinum electrodes.Preincubation with neuronal blocker, tetrodotoxin (TTX) was used todissect myogenic and neuronal components of contraction/relaxation.Preincubation with specific inhibitors were used to identify functionalneuronal subtypes: 1) nNOS-blocker Nω-Nitro-L-arginine methyl esterhydrochloride (L-NAME; 300 μM); and 2) VIP-receptor antagonist[D-p-Cl-Phe6, Leu17]-Vasoactive Intestinal Peptide (VIP-Ra; 2 μM).Following stimulation and subsequent contraction/relaxation andrecovery, tissues were washed with fresh medium, and allowed tore-establish a baseline before the next treatment. Equilibrated baselinewas arbitrarily set to zero, to measure contraction/relaxation due to astimulus.

Neurosphere Differentiation as a Function of Extracellular MatrixComposition

22×11 mm substrates were washed in Neutrad (Decon Labs, King of PrussiaPa.) and rinsed extensively in deionized water. Coverslips weresterilized by 70% ethanol, and subsequent UV exposure for 45 minutes.Coverslips were coated with poly-L-lysine (PLL; 1 mg/ml), PLL+10 μg/cm²type I collagen, PLL+10 μg/cm² type IV collagen or PLL+10 μg/cm²laminin. Composite coatings included:

-   -   5 μg/cm² Collagen I+5 μg/cm² type IV Collagen;    -   5 μg/cm² Collagen I+5 or 10 μg/cm² Laminin;    -   5 μg/cm² Collagen IV+5 or 10 μg/cm² Laminin;    -   5 μg/cm² Collagen I+5 μg/cm² Collagen IV+0.1 μg/cm² Heparan        Sulfate (HS);    -   5 μg/cm² Collagen I⁺5 μg/cm² Collagen IV+5 μg/cm² Laminin+0.1        μg/cm² HS.

Uncoated glass substrates were seeded with rabbit colonic smooth musclecells, and allowed to reach confluence. Rabbit enteric neurospheres wereharvested and treated with Accutase to obtain a mixture of single cellsas well as small neurospheres. 10,000 neuronal progenitor cells wereharvested and plated on to coated coverslips. To stimulatedifferentiation induced via soluble smooth muscle factors, each platewas shared by one confluent smooth muscle coverslip along with a coatedcoverslip containing adhered neurospheres. Enteric neurospheres wereallowed to differentiate for a period of fifteen days, with asupplementation of neuronal differentiation medium every 2 days(Neurobasal-A medium+1× B27 supplement+2% fetal calf serum+1×antibiotics).

Immunohistochemical Analysis of Neuronal and Glial Differentiation

Two time points were analyzed for neuronal and glial differentiation—day5 and day 15 post initiation of differentiation. Medium was aspiratedand cells on coverslips were fixed with 3.7 neutral bufferedformaldehyde. Cells were permeabilized with 0.15% Triton-X 100 andblocked with 10% horse serum. βIII tubulin was used to stain neuronalcells, and glial fibrillary acidic protein (GFAP) was used to stainglial cells. Primary antibodies were incubated for 1 hour at roomtemperature and unbound antibody was washed with PBS. Fluorophoreconjugated secondary antibodies (FITC-anti mouse and TRITC-anti rabbit)were used to visualize fluorescence using an inverted Nikon TiEfluorescent microscope. Staining with FITC-conjugated secondary antibodywithout the primary antibody was used as a negative control. Confluentsmooth muscle coverslips were stained with neuronal or glial markers toavoid a false positive staining while identifying differentiated neuronsor glia.

Data Analysis

Neurite lengths were measured from individual 10× micrographs obtainedat the same amplifier gain and exposure. Neurites were identifiedprimarily by expression of immunoreactivity for βIII tubulinconcurrently with neuronal morphology. Up to five sequential fields ofview were measured on each coverslip starting from one edge to theother, covering the area of the coverslip. All cells were measured oneach coverslip, covering the entire area of the neuronal coverslip. Thenumber of neurites measured for each substrate coating varied between20-50 readings. The length of the longest neurite from each cell wasmeasured using NIH Image J using the freeform tool. Neurite lengthsbetween coatings were compared using one way ANOVA, with Bonferronipost-test to identify a significant difference (p<0.05) in neuritelengths by varying culture substrata. GFAP immunofluorescence wasquantified using the Nikon Elements imaging software. Mean red (TRITC)fluorescence was calculated from 10× micrographs, using a constantrectangular area tool that covered 100% of the field of view. Multiple(at least 5) sequential fields of view at the same magnification werechosen for each sample to obtain mean fluorescence. One way ANOVA withBonferroni post-test was used to identify a significant difference inred fluorescent intensity between coated culture substrata. GraphPadPrism 5.1 for Windows (San Diego, Calif.) was used to performstatistical analysis. All statistics are from experiments between 3-5individual sets, with multiple micrographs within each set. Reportednumbers are mean±standard error of the mean. For neuronal subtypeanalysis, densitometry on western blots was performed using BioRadQuantity One (Hercules, Calif.). Raw data was acquired from the forcetransducer at 1000 samples/second. Second order Savitsky-Golay smoothingwas applied to data using GraphPad Prism 5.0 for Windows (GraphPadSoftware, San Diego, Calif.). Area under the curve (AUC) was measuredfrom the time of addition of pharmacological agonist/electrical field tothe end of the contraction/relaxation response. Extent of inhibition bypharmacological inhibitors was calculated by expressing the AUC ofcontraction/relaxation in the presence of the inhibitor as a percentageof the AUC in the absence of the inhibitor. One way ANOVAs with Tukeypost-tests were used to compare means using GraphPad Prism. p≤0.05 wasconsidered significant. Physiological evaluation and densitometry wascarried out between 3-5 tissue engineered sheets within eachexperimental set; all values are expressed as mean±SEM.

Neuroglial Differentiation Initial Phenotype of Rabbit EntericNeurospheres

Upon digestion of rabbit jejunal biopsies with dispase, near single cellsuspensions were obtained by filtration through 70 μm and 40 μm meshes.Single cells were approximately 7 μm in diameter. These cells wereplated in non-adherent culture dishes. Over the course of two weeks postplating, rabbit enteric neuronal progenitor cells aggregated andproliferated in culture and formed floating spherical structures, calledenteric neurospheres (FIG. 1A). Average neurospheres were 98.17±8.33 μm(n=34) two weeks post plating. The neurospheres continued to grow andaggregate, approaching 200-300 μm, whereupon they were broken down bytrituration. Upon immunohistochemical examination, the cells withinenteric neurospheres were positive for the low affinity nerve growthfactor receptor p75^(NTR) (FIG. 1B). They were additionally alsopositive for Sox2 (FIG. 1C, SRY related homeobox factor 2) and Nestin(FIG. 1D), a neuroepithelial stem cell marker. The results indicate thatneurospheres derived from the rabbit intestine following this procedurecontained neural-crest derived cells, capable of differentiation in toenteric neurons and/or enteric glia.

Neuronal progenitor cells were isolated from full thickness biopsies ofadult rabbit jejunums that aggregate in culture to form floatingspherical colonies, dubbed enteric neurospheres (FIG. 1A). The entericneurospheres were comprised of cells positive for p75, Sox2 and Nestin(FIG. 1 B-D). The presence of p75^(NTR) confirms the neural-crestlineage of the isolated cells. The presence of Sox2 and Nestin confirmsthe progenitor status of the isolated cells, indicating that these cellsare similar to enteric neuronal progenitor cells previously isolatedfrom the gut that have the potential to differentiate into both neuronsand glia.

Neuronal Differentiation on Individual ECM Substrates (Collagen I,Collagen IV or Laminin)

Poly-L-lysine (PLL) coating was a pre-requisite to enteric neurosphereadhesion to glass substrates. Glass coverslips that lacked any coatingdid not support enteric neurosphere adhesion sufficiently todifferentiate into neurons or glia. In order to maintain uniformity, allcoverslips were initially coated with PLL and additionally with laminin,collagen I or collagen IV. All coated coverslips required between 2-4hours for enteric neurospheres to attach.

Enteric neurospheres on coated coverslips were allowed to differentiateinitially using neuronal differentiation medium alone. However, severalsets of experiments demonstrated no morphological evidence ofdifferentiation at the day 15 timepoint. Thereby, in order to render thesoluble environment conducive to differentiation, a confluent coverslipcontaining colonic smooth muscle cells was placed in the same culturedish (FIG. 2). The neuronal coverslip (coated with ECM substrate andcontaining enteric neurospheres) and the smooth muscle coverslipsthereby shared soluble factors. The addition of the smooth musclecoverslip marked the initiation of differentiation (day 0).

Morphological evidence of neuronal or glial differentiation was readilyvisible by day 5. A later time point (day 15) was identified to studythe development of mature neurons or glia in vitro as a function of ECMcomposition. During the differentiation process, the culture dishesremained undisturbed till the early time point (day 5) or the late timepoint (day 15), except for medium supplementation. Neuronaldifferentiation was identified by immunofluorescent staining of theneuronal coverslip at either day 5 or day 15 with an antibody directedagainst βIII Tubulin.

Day 5 Timepoint:

Even in the presence of smooth muscle, enteric neurospheres on PLLremained undifferentiated, with some progenitor cells withinneurospheres expressing low levels of βIII tubulin (FIG. 3A). However,with the addition of laminin, collagen I or collagen IV to PLL on theculture substrata, neuronal differentiation was evident by day 5 (FIG.3B-D). Neurite lengths varied non-significantly between 193 μm and 288μm on ECM substrata at the early time point (FIG. 9A). Neurons oncollagen IV and laminin coated coverslips demonstrated a higher level ofbranching (two or more neurites per cell; FIG. 3B,D).

Neurospheres and neuronal progenitor cells attached to the PLL coatedcoverslips, and stayed attached at day 5, but did not initiate neuronaldifferentiation. However, glial differentiation was readily visible byday 5, and improved by day 15 (FIG. 3A,E; FIG. 6A,E). Entericneurospheres on PLL substrates indicated a preference towards glialdifferentiation versus neuronal differentiation.

Day 15 Timepoint:

At the day 15 timepoint, neurospheres on PLL coverslips barely initiatedneuronal differentiation, evidenced by a flatter morphology and theappearances of faint tubulin-positive extensions (FIG. 3E). With theaddition of laminin, collagen I or collagen IV, neurite lengths weresignificantly longer compared to PLL (p<0.001). Differentiated neuronson laminin coverslips demonstrated the longest neurite extensions(326.9±13.25 μm, n=27, FIG. 3F), significantly longer than collagen I orcollagen IV (p<0.05, FIG. 9A). By day 15, neurons on collagen I-coatedcoverslips still had no significant branching compared to those oncollagen IV-coated coverslips (FIGS. 3G-H).

Neuronal Differentiation on Collagen Laminin Substrates

In the next set of experiments, combinations of collagens and lamininwere evaluated. Two concentrations of laminin were evaluated to identifythe minimum amount of laminin required to influence neuronaldifferentiation. Coverslips were coated with either collagen I orcollagen IV with 5 μg/cm² or 10 μg/cm² of laminin. The addition oflaminin enhanced neuronal differentiation when compared to individualcollagen substrates (compare FIG. 4 with FIG. 3), but no significantdifference was observed in neurite length between the two concentrationsof laminin.

Collagen I and Laminin:

At the day 5 timepoint, addition of laminin to collagen I increased thenumber of progenitor cells undergoing neuronal differentiation, but didnot alter neuronal branching or neurite lengths significantly (FIG.4A,B,E,F). At the day 15 timepoint, significantly (p<0.05) enhancedneuronal differentiation was observed compared to collagen I. Neuritelengths on collagen-laminin substrates at day 15 (280 μm-290 μm;n=27-34) were longer than collagen I substrates (235.5±10.05 μm; FIG.9B).

Collagen IV and Laminin:

The addition of laminin to collagen IV enhanced neuronal differentiationwhen compared to coverslips coated individually with collagen IV only(compare FIG. 4C,D,G,H to FIG. 3D,H). At the day 5 timepoint, theaddition of laminin increased the number of cells undergoing neuronaldifferentiation. No significant difference was observed in neuritelengths at day 5 (247 μm-288 μm; FIG. 9B). At the day 15 time point,coverslips coated with both collagen IV and laminin had significantly(p<0.05) longer neurites (324 μm compared to 281 μm, FIG. 4G-H).Initiation of inter-neuronal networking was also observed. There was noobservable or significant difference in neurite lengths between the twoconcentrations of laminin used (5 μg/cm² or 10 μg/cm²; FIG. 9B).

Neuronal Differentiation on Composite ECM Substrates with Laiminin andHeparan Sulfate

In this additional set, the effect of a combination of collagens onneuronal differentiation was investigated. Composite coatings wereevaluated with a 2:1 mix of Collagen I/Collagen IV as the base. Thiscomposite collagen base was evaluated first. Additionally, neuronaldifferentiation was evaluated on substrates that included laminin and/orheparan sulfate in combination with composite collagen.

Heparan sulfate interacted with both collagen IV and with laminin topositively modulate neuronal differentiation, evidenced by the enhancedneurite lengths and initiation of neuronal networking (FIG. 4 A-H).Composite collagen substrates with laminin and/or heparan sulfate allmaintained a low level of GFAP positive glial cells, with initiation ofastrocytic networking becoming more obvious at the later time point.

Composite Collagen I/Collagen IV:

Several cells underwent neuronal differentiation (FIG. 5A, E; similar toindividual coatings of either collagen I or collagen IV), but neuritelengths were significantly shorter on composite collagen substrates atday 5. Neurite lengths measured at 156±7.23 μm on day 5. At day 5,neuronal differentiation progressed and individual neurons had multiplebranches and long neurites (FIG. 5E). Neurite lengths averaged at241.2±9.387 μm on day 15 (FIG. 9C).

Addition of Laminin:

Addition of laminin to composite collagen substrates increased thenumber of differentiated neurons visible by day 5 (FIG. 5B). Neuritelengths were significantly (p<0.05) longer on substrates containinglaminin (215±7.57 μm; n=43). At the day 15 time point, substratescontaining composite collagen and laminin demonstrated significantclustering of neurons (FIG. 5F), with an additional 71 μm increase inneurite length, averaging at 286.8±9.521 μm, n=50.

Addition of Heparan Sulfate:

Addition of heparan sulfate also dramatically increased the number ofprogenitor cells undergoing neuronal differentiation by day 5 (FIG. 5C).Average neurite lengths on substrates containing heparan sulfate alongwith composite collagen was 212.8±9.46 μm; n=43 at day 5. At day 15, theinitiation of neuronal networking was visible with βIII Tubulin staining(FIG. 5G).

Addition of Laminin and Heparan Sulfate:

The addition of laminin and heparan sulfate together with the compositecollagen increased the number of differentiated neurons as well as thelength of the individual neuronal processes and neurite branching (FIG.5D). At day 15, initiation of neuronal networking with significantclustering of neurons was observed (FIG. 5H). Neurite lengths weresignificantly longer (325±19.37 μm) compared to composite collagen alone(FIG. 9C).

Glial Differentiation on Individual ECM Coatings (Collagen I, CollagenIV or Laminin)

In addition to neuronal differentiation studies described above, glialdifferentiation was also studied as a function of ECM composition ofculture substrata. Enteric neurospheres were plated on to coatedcoverslips in duplicate, and one coverslip was used to evaluate neuronaldifferentiation while a duplicate coverslip was used to evaluate glialdifferentiation. A primary antibody directed against Glial fibrillaryacidic protein (GFAP) was utilized to identify glial differentiation.Fluorescent microscopy was used to visualize differentiated glia, usinga TRITC fluorophore conjugated secondary antibody. The Nikondocumentation software was used to calculate mean red fluorescenceindicating the number of differentiated glia in a field of view ofconstant area.

The presence of several axolemmal fragments can arrest the proliferationof glia. This is in line with the low levels of GFAP immunofluorescenceobserved on substrates that supported extensive neuronaldifferentiation. The only substrates that supported differentiation ofenteric neuroglial progenitor cells into glia extensively were PLL andindividual coatings of collagen I/IV. Neuronal differentiation waspresent on these substrates, but not as extensively as any of the othercomposite coatings that included laminin and heparan sulfate.

Day 5 Timepoint:

In the presence of smooth muscle, enteric neurospheres on PLL coatedcoverslips demonstrated significant GFAP staining by day 5 (15.29±1.29AU; FIG. 6A). In contrast, enteric neurospheres on PLL coverslips didnot demonstrate significant neuronal differentiation at day 5,indicating the preferential differentiation in to glia at the early timepoint on PLL coverslips. With the addition of laminin, entericneurospheres demonstrated highly significantly reduced GFAP staining(0.3825±0.2 AU). Undifferentiated neurospheres on the laminin coverslipscontained several progenitor cells that were positive for GFAP (FIG.6B). On the same laminin coated coverslips, neuronal differentiation wasextensive at the early timepoint, indicating an early preference forneuronal differentiation in the presence of laminin (FIG. 3B). Minimalglial differentiation was observed on either of the collagen substrates(FIG. 6C-D).

Day 15 Timepoint:

By the late day 15 time point, PLL coated coverslips had the highestnumber of glia, indicated by a highly significant (p<0.0001) GFAPfluorescent intensity, averaging at 28.56±1.14 AU (FIG. 6E, 9D). Gliawere apparent on ECM-coated coverslips as well, but to a lower extentthan on PLL. In contrast to day 5, laminin coated coverslipsdemonstrated the presence of several glia at the day 15 time point and arobust GFAP fluorescent intensity was observed (FIG. 5F, 16.54±0.32 AU).Several glia were observed by day 15 on each of the collagen substrates,with fluorescence ranging from 11.84 to 13.38 AU. (FIGS. 6G-H).

Glial Differentiation on Collagen-Laminin Substrates

Similar to neuronal differentiation, glial differentiation was evaluatedon substrates that were coated with either collagen I or cCollagen IVwith laminin. The addition of laminin to collagen coated coverslips didnot inhibit glial differentiation. Several differentiated glia wereobserved on day 5 (8.4±0.75-14.08±0.3 AU) on collagen-laminin substrates(FIG. 7A-D). There was no significant difference in the number of GFAPpositive cells at the early time point with the addition of laminin (5μg/cm² or 10 μg/cm²) to either collagen I or collagen IV substrates FIG.9D). Robust GFAP expression (12.6±1.29-14.22±1.01 AU) was observed atthe day 15 time point on all collagen-laminin substrates, notsignificantly different from one another (FIG. 7E-H).

Glial Differentiation on Composite ECM Substrates with Laminin andHeparan Sulfate

Glial differentiation was evaluated by varying the culture substratumwith a combination of collagen I and IV. Additionally, the effect of theaddition of laminin and/or heparan sulfate was also studied on glialdifferentiation.

Composite Collagen I/Collagen IV:

Glial differentiation peaked on day 5, on coverslips coated with thecollagen I/IV mixture (FIG. 8A). Red fluorescence (15.75±0.49 AU) wascomparable to that on PLL coated coverslips at day 5 (FIG. 9D). Incontrast, neuronal differentiation on composite collagen coatedcoverslips was poor at the early time point, indicating a preferentialdifferentiation into glia early on. By day 15, initiation of clusteringof glial cells was observable (FIG. 8E), with no significant increase inred fluorescence.

Addition of Laminin and/or Heparan Sulfate:

Early glial differentiation at day 5 was significantly reduced(10.16±0.8 to 11.06±0.5) with the addition of laminin and/or heparansulfate to composite collagen substrates (FIG. 8B-D). In contrast, thesesubstrates supported neuronal differentiation extensively (compare FIG.8A-D to FIG. 5A-D), indicating a preferential neuronal differentiationat the early time point. At the later day 15 time point, anon-significant increase in the number of glia and thereby increase inred fluorescence was observed (FIG. 8E-H, FIG. 9D).

Neuronal Subtype Differentiation

Ultrastructure and Viscoelastic Properties of ECM Hydrogels:

All compositions of ECM hydrogels gelled at 37° C. within 30 minutes.Scanning electron micrographs revealed a fibrous structure in type ICollagen gels (FIG. 10A). The fibers were randomly oriented, withdiameters averaging at 478.3±19.31 μm. With the addition of type IVCollagen, network-like structures were observed (FIG. 10B). Cables offibers within the networked structures were thicker, with averagediameters of 714.8±36.67 μm. Addition of laminin to the hydrogels didnot alter the ultrastructure or the networked suprastructure (FIG. 10C).With the addition of heparan sulfate, the fibers within the networkedstructures were pulled more tightly together and cabled (FIG. 10D). Thedehydrated ECM gels displayed a porous appearance, with average porosityranging from 40.77%-43.95% (FIG. 1, table).

Viscoelastic moduli were measured in hydrated ECM gels using oscillatoryrheometry. Type I Collagen gels had increasing viscoelastic moduli withincreasing collagen concentration ranging from 72.6±4.86 Pa (800 μm/ml)to 182.3±2.6 (1600 μm/ml) to 424±2 Pa (3200 μm/ml). The addition of 200μm/ml collagen IV to 800 μm/ml collagen I increased the modulus of thegels to 236±13.53 Pa. The addition of laminin had no effect onviscoelastic moduli (compare 236±13.53 Pa to 220.7±16.27 Pa). 10 μm/mlof heparan sulfate caused an increase in the modulus of ECM hydrogels(287±20.11 Pa, p<0.05). FIG. 1 (table) summarizes that the final ECMgels evaluated had viscoelastic moduli ranging from 182 Pa to 287 Pa.

Neuronal Differentiation in Engineered Innervated Intestinal SmoothMuscle Sheets:

Uniaxially-aligned smooth muscle cells compacted overlaying ECMhydrogels over 10 days in culture as described before. The resultanttissue engineered sheets were ˜1 cm long, and a few cell layers thick.In the presence of smooth muscle, the enteric neuronal progenitor cellsdifferentiated within the ECM hydrogel. Neuronal differentiation wasidentified morphologically by microscopic examination at day 10,demonstrating similar differentiation profiles expressed by entericneuronal progenitor cells, both in vitro and in tissue engineeredconstructs. Several differentiated neurons were observed in tissueengineered sheets, regardless of the ECM composition (FIG. 11). Arrowsin the figures indicate numerous instances of neuronal clustering andpreliminary neuronal networking.

Neuronal Composition in Engineered Innervated Intestinal Smooth MuscleSheets:

Immunoblotting was used to assess neuronal composition within tissueengineered innervated intestinal smooth muscle sheets. Blotting forβ-actin demonstrated that equal amounts of protein were assayed.Representative blots for each protein are shown, indicating theapproximate molecular weight at which they appear on the gels (FIG.12E). Contractile phenotype of constituent smooth muscle wasdemonstrated by the similar expression of smoothelin, within the tissueengineered sheets (FIG. 12B). The expression of Smoothelin was constant,regardless of the ECM composition of the sheets, indicating that theconstituent smooth muscle cells maintained a contractile phenotype.

Neuronal Differentiation:

Pan neuronal marker βIII Tubulin expression was similar amongst alltissue engineered sheets, despite the ECM composition (FIG. 12A). Thissuggested that irrespective of the ECM composition, neuronaldifferentiation of enteric neurospheres proceeded similarly in thepresence of smooth muscle cells. βIII Tubulin expression ranged from21.65±1.43 AU-28.98±0.85 AU. βIII Tubulin expression was similar amongstvarious ECM gel compositions (ns; FIG. 12A), indicating similar neuronaldifferentiation.

Cholinergic Neurons:

Choline acetyltransferase (ChAT) expression was used to detect thepresence of cholinergic neurons (FIG. 12C). Collagen I (33.73±1.13 AU)and collagen I/IV/laminin (28.82±1.21 AU) sheets had a significantlyelevated expression of ChAT compared to sheets with composite collagenand/or heparan sulfate. Immunoblotting demonstrated an enrichedcholinergic neuron population in tissue engineered sheets manufacturedwith collagen I only or composite collagen I/IV with laminin. Thepresence of cholinergic neurons was additionally confirmed usingimmunohistochemistry (FIG. 13E-H).

Nitrergic Inhibitory Motor Neurons:

Neuronal nitric oxide synthase (nNOS) expression was used to detect thepresence of inhibitory nitrergic motor neurons (FIG. 12D). Sheets withcollagen IV (with or without laminin/heparan sulfate) had asignificantly higher nNOS expression ranging from 26.37±1.29AU-28.15±2.69 AU. Conversely to ChAT, collagen I sheets had minimal nNOSexpression (11.33±2.85 AU). Presence of nNOS was additionally confirmedusing immunohistochemistry (FIG. 13I-L).

VIP-Ergic Inhibitory Motor Neurons:

Vasoactive intestinal peptide (VIP) motor neurons were identified usingimmunohistochemistry. VIP neurons were abundant, with increasedimmunofluorescence in composite hydrogels with laminin and heparansulfate (FIG. 13A-D).

Substrates that supported peptidergic neuron differentiation may resultin enriched populations of peptidergic neurons comprising greater than25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,100%, or any intermediate percentage.

Agonist Induced Contractility of Tissue Engineered Innervated SmoothMuscle Sheets

Potassium Chloride-Induced Contraction:

Electromechanical coupling integrity of constituent smooth muscle cellswas first evaluated using potassium chloride (KCl). KCl treatmentelicited rapid contractions that were sustained for ˜5 minutes (FIG.14). Peak maximal contraction in response to KCl was similar between thedifferent tissue engineered sheets (FIG. 14A-D), ranging from 279.5±4.79μN to 296.5±6.26 μN. This correlated with the equivalent expression ofcontractile smooth muscle marker, Smoothelin, indicating that theconstituent smooth muscle cells within the tissue engineered sheetsmaintained a contractile phenotype regardless of ECM composition.Furthermore, KCl-induced contractions in tissue engineered sheets weresimilar to native rabbit intestinal tissues (FIG. 14E) in time course,but slightly reduced in magnitude. Peak KCl-induced contractions innative tissue averaged 373.5±10.63 μN. KCl-induced contraction wasunaffected by pre-treatment with neuronal blocker TTX (grey traces; FIG.14), indicating myogenic electromechanical coupling integrity. FIG. 16Fdemonstrates that the area under the curve of contraction was similar inall tissue engineered sheets, and was significantly higher in nativerabbit intestinal tissues. Although reduced in magnitude compared tonative tissue, KCl-induced contractions were similar among the differenttissue engineered sheets, indicating a robust contractile smooth musclephenotype unaffected by the ECM composition.

Acetylcholine-Induced Contraction:

Exogenous addition of 1 μM Acetylcholine (Ach) was used to simulateagonist-induced contraction. All tissue engineered sheets contracted inresponse to Ach, and sustained contractions up to ˜5 minutes poststimulation with Ach (FIG. 15A-D). Tissue engineered sheets withcomposite collagen I/IV with laminin had a significantly elevated peakmaximal Ach-induced contraction (FIG. 15C; 232.9±8.167 μN), as well asan elevated area under the curve of contraction (47606±2054 AU).Magnitude of Ach-induced contraction was still significantly lowercompared to contraction in native tissue (342.6±3.15 μN; 70448±5876 AU).However, the time course of contraction was very similar to nativetissue in tissue engineered sheets containing laminin, reaching maximalcontraction within a minute of agonist stimulation. Collagen I sheetsalso had an elevated Ach-induced contraction (FIG. 15A; 238.9±13.72 μN;42668±2172 AU) corresponding to the elevated ChAT protein expression.However, the kinetics of contraction did not match native tissue.

In order to estimate the smooth muscle (myogenic) component ofAch-induced contraction, neurotoxin TTX was used as a pretreatment (greytraces, FIG. 15). Area under the curves of contraction was compared withand without TTX pre-treatment in order to estimate % inhibition (FIG.15F). Percent inhibition of Ach-induced contraction in the presence ofTTX was highest in two ECM conditions: i) collagen I sheets(72.77±2.45%); and ii) collagen I/IV/laminin sheets (60.58±1.66%). Thesevalues of % inhibition were similar to that observed in native tissue(72.73±3.66%) upon TTX-pretreatment. This increased neuronalcontribution to Ach-induced contraction also correlated with theelevated protein expression of ChAT in collagen I and composite collagenI/IV/laminin sheets (FIG. 12E, G). TTX-pretreatment inhibitedAch-induced contraction to a significantly lower extent in collagenI/IV±heparan sulfate sheets, ranging from 48.36±4.36% (Heparan sulfate)to 50.31±4.22% (collagen IV; FIG. 15F).

Relaxation in Engineered Innervated Sheets in Response to ElectricalField Stimulation:

Electrical field stimulation (EFS) at 5 Hz, 0.5 ms was used to stimulateneurons within the tissue engineered sheets to produce relaxation ofsmooth muscle (FIG. 16). The extent of relaxation was quantified as areaunder the curve of relaxation. Extent of relaxation significantly variedamongst the tissue engineered sheets with varying ECM compositions.Sheets bioengineered with collagen IV, which displayed elevated nNOSexpression, had higher relaxation compared to sheets bioengineered withcollagen I only (compare 109693±8465 AU in collagen I/IV sheets to23142±4921 in collagen I sheets). Sheets containing laminin and/orheparan sulfate also had significantly elevated relaxation compared tocollagen I sheets (68395-69025 AU). In response to EFS, native tissuesrelaxed generating 101550±11279 AU. Tissue engineered sheets withcollagen IV and/or laminin and/or heparan sulfate additionally had atime course of relaxation most similar to native tissue. Maximalrelaxation was achieved within 2 minutes of EFS, and a subsequentrecovery of basal force was complete within 10 minutes. Uponpre-treatment with TTX, EFS-induced relaxation was inhibited entirely(grey traces, FIG. 16).

Inhibition of Nitric Oxide Synthase:

In order to identify the presence and functionality of nitrergicneurons, an inhibitor of nitric oxide synthase (L-NAME) was used (greytraces, FIG. 17). Percent inhibition was determined by comparing areasunder the curves of maximal relaxation with and without the L-NAMEpre-treatment. Percent inhibition with L-NAME treatment was the lowestin collagen I sheets (33.37±8.37%; grey trace, FIG. 17A). Thiscorresponded to the low protein expression of nNOS in collagen I sheetscompared to sheets containing collagen IV (FIG. 12D). In contrast, theinhibition of nNOS activity attenuated relaxation up to 61.71±2.82%(grey trace, FIG. 17B) in collagen I/IV sheets. In sheets containinglaminin and heparan sulfate, % inhibition with L-NAME varied between62.28±2.75% (laminin, FIG. 17C) to 57.16±1.91% (heparan sulfate). Thisinhibition is significantly elevated compared to collagen I sheets,corresponding to the increased expression of nNOS observed in thecollagen I/collagen IV sheets (FIG. 12C). Native tissues had a higher %inhibition with L-NAME (78.02±2.85%).

Inhibition of the VIP-Receptor:

The functionality of VIP-ergic neurons was assessed using a VIP receptorantagonist (VIP-Ra). Pre-treatment with VIP-Ra inhibited maximalrelaxation in all tissue engineered sheets to varying extents rangingfrom 55.55±3.92%-65.92±5.38% (grey traces, FIG. 18). Inhibition ofEFS-induced relaxation indicated the presence of differentiatedVIP-ergic neurons in tissue engineered sheets.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting. While the applicants' teachingsare described in conjunction with various embodiments, it is notintended that the applicants' teachings be limited to such embodiments.On the contrary, the applicants' teachings encompass variousalternatives, modifications, and equivalents, as will be appreciated bythose of skill in the art.

1-13. (canceled)
 14. A method of preparing an innervated smooth muscleconstruct, the method comprising: obtaining a population of longitudinalsmooth muscle cells; culturing the longitudinal smooth muscle cells toform a uniaxially-aligned smooth muscle sheet; obtaining a population ofneural stem cells; and co-culturing the neural stem cells andlongitudinal smooth muscle cells on a substrate comprising at least oneextracellular matrix (ECM) material component.
 15. (canceled)
 16. Themethod of claim 14 wherein the step of culturing the longitudinal smoothmuscle cells further comprises culturing the muscle cells on a moldcontaining a wavy microtopography to form the uniaxially-aligned smoothmuscle sheet.
 17. The method of claim 14 wherein the substrate furthercomprises a hydrogel.
 18. The method of claim 17 wherein the hydrogelcomprises at least one of collagen, laminin and heparan sulfate.
 19. Themethod of claim 17 wherein the hydrogel comprises collagen.
 20. Themethod of claim 17 wherein the hydrogel comprises at least 800 μg/ml ofcollagen type I.
 21. The method of claim 20, wherein the hydrogelcomprises between about 800 μg/ml and about 1600 μm/ml collagen I. 22.The method of claim 17 wherein the hydrogel further comprises at least200 μg/ml of collagen type IV.
 23. The method of claim 17 wherein thehydrogel further comprises at least 5 μg/ml of laminin.
 24. The methodof claim 17 wherein the hydrogel is substantially free of laminin. 25.The method of claim 17 wherein the hydrogel is substantially free ofheparan sulfate.
 26. The method of claim 14 wherein the method furthercomprises administering the construct to a patient.
 27. The method ofclaim 26, wherein the step of administering the construct furthercomprises implanting the construct into the patient.
 28. The method ofclaim 27 wherein the construct comprises a hydrogel sponge witheffective pore sizes ranging from 10 nanometers to 10 micrometers.